The increased shortage of donor organs has sparked a world-wide interest in xenotransplantation, i.e., the replacement of human organs or tissues with those from a donor of a different species, such as pigs. Recent progress offers cause for optimism, but there are obstacles that must be overcome.
Xenotransplants have been classified into two groups, concordant and disconcordant, based on the phylogenetic distance between species. Animals that are evolutionarily close and do not have natural antibodies specific for each other are termed concordant. Animals that are phylogenetically distant and reject organs in a hyperacute manner are termed discordant. There are many gradations in between and exceptions to the rule.
Non-human primates would be the logical source of organs for humans, in that they are most closely related. However, due to considerations of size of the organ, lack of availability, and the likelihood of transmission of infectious diseases, most researchers have determined that primates are not a preferred source of organs. Instead, the swine is the likely choice for a source of organs, because of its ready availability, similarity in organ size, its breeding characteristics, and the similarity of its organ systems to humans. However, the swine is a discordant species to humans.
Xenografts are subject to all four rejection mechanisms: (1) hyperacute rejection mediated by preformed antibodies, (2) early or accelerated rejection mediated by induced antibodies, (3) delayed xenograft rejection or acute vascular rejection (DXR/AVR) mediated by T-cells, and (4) chronic rejection mediated by B-cell and T-cell mechanisms. Induction of all four mechanisms can be attributed to a greater number of foreign antigens present than in an allograft tissue (one from the same species, i.e., human). Further, human inhibitory receptors often do not interact with the other species"" class I MHC molecules, thus allowing the activation of various rejection mechanisms that proceed uninhibited. Transplantation of porcine pancreatic islets and of a pig liver into human patients has been reported, (Makowka, et al., 1993; Satake, et al., 1993; Tibell, et al., 1993), but the outcomes were not positive. Improved inhibition of transplant rejection with drug therapy may lead to better outcomes.
Hyperacute rejection of xenografts is initiated by the binding of xenoreactive antibodies to donor endothelial cells followed by the activation of complement, predominaritly via the classical pathway. Pigs, for example, express an endothelial carbohydrate determinant, gal xcex1 (1,3) gal, that is not expressed in humans, and is considered a new blood group antigen to the human immune system. Complement activation induces type I activation of the endothelial cells, a process which is rapid and independent of protein synthesis. It is characterized by reversible cell retraction, translocation of P-selectin to the apical surface, and the elaboration of a variety of vasoactive substances. Furthermore, heparin sulphate proteoglycans are released from the endothelial cell surface leaving the cell susceptible to procoagulant and complement-mediated injury. Critical functions of endothelial cells are lost, and the end-result is interstitial hemorrhage, diffuse thrombosis, and irreversible organ damage occurring from within minutes to several hours following transplant. These are the characteristic features of HAR. HAR can be prevented by reducing either complement activity or the level of naturally occurring anti-xenograft antibodies.
Even if one reduces or eliminates HAR, the xenograft will be rejected after a few days by the process designated delayed xenograft rejection or acute vascular rejection (DXR/AVR). DXR/AVR is characterized by type II endothelial cell activation, which is protein synthesis dependent. Although DXR/AVR is thought to be largely complement independent, some studies indicate that complement may still be involved in DXR/AVR. Inhibition of complement by soluble complement receptor type I (sCR1) combined with immunosuppression delayed the occurrence of DXR/AVR of porcine hearts transplanted into cynomolgus monkeys (Davis, EA et al., Transplantation 62:1018-23 (1996)). Transplantation of pig kidneys expressing human decay accelerating factor to cynomolgus monkeys also had some protective effect against DXR/AVR (Zaidi A et al. Transplantation 65:1584-90 (1998); Loss M et al., Xenotransplantation 7: 186-9(2000)). Addition of anti-endothelial cell antibodies and complement in sublytic doses induced expression of tissue factor (Saadi S et al., J. Exp. Med. 182:1807-14 (1995)). Porcine endothelial cells exposed to human serum expressed plasminogen activator inhibitor (Kalady M F et al., Mol. Med. 4:629-37 (1998)) and increased the expression of various chemokine genes (Selvan RS et al., J. Immunol. 161:4388-95 (1998)). The increased expression of various chemokine genes was found to be complement dependent. Nevertheless, an anti-C5 monoclonal antibody was shown to be effective in preventing HAR, but not DXR/AVR (Wang, H. et al., Transplantation 68:1643-51 (1999)).
E-selectin (also known as ELAM-1, CD62, and CD62E) is a cytokine inducible cell surface glycoprotein cell adhesion molecule that is found exclusively on endothelial cells. E-selectin mediates the adhesion of various leukocytes, including neutrophils, monocytes, eosinophils, natural killer (NK) cells, and a subset of T cells, to activated endothelium (Bevilacqua, et al., Science 243: 1160 (1989); Shimuzu, et al., Nature 349:799 (1991); Graber, et al., J. Immunol. 145: 819 (1990); Carlos, et al., Blood 77: 2266 (1991); Hakkert, et al., Blood 78:2721 (1991); and Picker, et al., Nature 349:796 (1991)). The expression of E-selectin is induced on human endothelium in response to the cytokines IL-1 and TNF, as well as bacterial lipopolysaccharide (LPS), through transcriptional upregulation (Montgomery, et al., Proc Natl Acad Sci 88:6523 (1991)).
The human leukocyte receptor for human E-selectin has been identified (Berg, et al., J. Biol. Chem. 23: 14869 (1991) and Tyrrell, et al., Proc Natl Acad Sci 88:10372 (1991)). Structurally, E-selectin belongs to a family of adhesion molecules termed xe2x80x9cselectingxe2x80x9d that also includes P-selectin and L-selectin (see reviews in Lasky, Science 258:964 (1992) and Bevilacqua and Nelson, J. Clin. Invest. 91:379 (1993)). These molecules are characterized by common structural features such as an amino-terminal lectin-like domain, an epidermal growth factor (EGF) domain, and a discrete number of complement repeat modules (approximately 60 amino acids each) similar to those found in certain complement binding proteins.
Clinically, increased E-selectin expression on endothelium is associated with a variety of acute and chronic leukocyte-mediated inflammatory reactions including allograft rejection (Allen, et al., Circulation 88: 243 (1993); Brockmeyer, et al., Transplantation 55:610 (1993); Ferran, et al Transplantation 55:605 (1993); and Taylor, et al., Transplantation 54: 451 (1992)). Studies in which the expression of human E-selectin in cardiac and renal allografts undergoing acute cellular rejection was investigated have demonstrated that E-selectin expression is selectively upregulated in vascular endothelium of renal and cardiac tissue during acute rejection. Id. Additionally, increased E-selectin expression correlates with the early course of cellular rejection and corresponds to the migration of inflammatory cells into the graft tissue. Id. Taken together, these studies provide evidence that cytokine-induced expression of E-selectin by donor organ endothelium contributes to the binding and subsequent transmigration of inflammatory cells into the graft tissue and thereby plays an important role in acute cellular allograft rejection.
Blocking the upregulation of E-selectin, which is a major hallmark for type II endothelial cell activation characteristic of DXR/AVR, would be a potential strategy to treat and prevent DXR/AVR.
The invention includes C5 inhibitors that bind to C5 and inhibit type II endothelial cell activation, as well as suppressing the upregulation of E-selectin on endothelial cells. These C5 inhibitors are useful for the treatment and prevention of xenograft rejection, and in particular DXR/AVR. The C5 inhibitors may also inhibit the formation of C5a, inhibit the formation of Terminal Complement Complex (xe2x80x9cTCCxe2x80x9d) and/or block complement mediated cell lysis. The inhibitors include monoclonal antibodies (xe2x80x9cMAbxe2x80x9d) as well as homologues, analogues and modified or derived forms thereof, including immunoglobulin fragments like Fab, F(abxe2x80x2)2, Fv and single chain antibodies. Small molecules including peptides, oligonucleotides, peptidomimetics, and organic compounds with the same functional activity are also included.
One example of a MAb which bound to C5 was shown, in an in vitro model, to be useful in treatment of xenograft rejection and DXR/AVR, was generated as described below and designated 137-76. Other examples include the Anti-C5 MAbs 137-10, 137-21, 137-30, and 137-50. The invention also includes monoclonal antibodies that bind to the same epitope as either MAb 137-76 or MAb 137-30.
The treatment of delayed xenograft rejection or acute vascular rejection involves the administration of a C5 inhibitor of the invention that inhibits type II endothelial cell activation and which can be manifested by the suppression of E-selectin expression.